Examining Skeletal Stem Cell Diversity and its Role in Intervertebral Disc Regeneration

项目摘要/摘要。 椎间盘(IVD)是一种高度专业化的纤维软骨结构，其恶化速度更快。 比身体里的任何其他结缔组织都要多。这种情况通常被称为IVD变性和 IVD修复的关键挑战是开发有效的治疗方法来逆转纤维软骨 损坏。由于其细胞固有的自我更新和分化特性，组织居留利用 干细胞有望成为对抗IVD变性的一种基于干细胞的方法。我们的实验室是第一个 真性骨骼干细胞(SSCs)的鉴定、分离及功能鉴定 在小鼠和人类中严格产生骨、软骨和骨髓基质的下游祖细胞32，33。 干细胞与间充质基质细胞不同，间充质基质细胞代表高度不同的细胞类型的混合物57。 自那以后，我们利用我们对SSCs的知识来了解退行性骨骼疾病，包括 骨关节炎、骨不连和年龄相关性骨质疏松症46、58、59。最近，我们使用我们的隔离方法 在小鼠和人类IVD中发现组织驻留的SSCs。在这项提案中，总体目标是 确定决定SSC进入IVD组织命运的内在线索，并检查可能 使用一种新的小鼠尾部IVD微骨折手术模型指导IVD再生。我们的预赛 数据表明，IVD SSCs在分化能力上明显比股骨SSCs更具软骨形成能力 在体外和体内都有。此外，我们还发现，小鼠IVD SSCs只表达HOXA4及其受体 骨骼命运的决定可以通过在体外添加形态因子FGF2、SHH和WNT3a来决定。我们也 发现小鼠尾部IVDS的急性微骨折损伤不会放大驻留的IVD SSCs和 将IVD SSCs移植到IVD的微骨折中不能产生纤维软骨，因此表明它们可能 体内软骨分化需要额外因素的指导。在这项提案中，我们的总体 假设HOXA4是内在调节因子，而FGF2是IVD组织命运的外在调节因子， 在IVDSSCs中调节这些通路可以作为一种潜在的干细胞靶向疗法 对抗IVD退行性疾病。在目标1中，我们将通过调节HOXA4的表达来解决这一假设 通过慢病毒转导的IVD和股骨(对照)SSCs，随后在体外和体内执行 分化实验评估其软骨细胞的IVD产量。在目标2中，我们将测试注射形态生物质是否 FGF2、SHH和WNT3a可改变微骨折内SSCs再生损伤的命运决策 体内IVD组织。最终，这套基础和临床前拟议的实验将进一步定义 SSC多样性的概念，并为干细胞治疗的临床翻译奠定了基础 预防和逆转与IVD相关的肌肉骨骼疾病。

Project Summary/Abstract. The intervertebral disc (IVD) is a highly specialized, fibrocartilaginous structure that deteriorates at a rate faster than any other connective tissue in the body. This condition is commonly referred to as IVD degeneration and a critical challenge for IVD repair is the development of effective treatments that reverses the fibrocartilage damage. Due to their cell intrinsic properties of self-renewal and differentiation, the utilization of tissue resident stem cells holds promise as a stem cell-based approach to combat IVD degeneration. Our lab was the first to identify, isolate and functionally characterize bona fide skeletal stem cells (SSCs) and their committed downstream progenitors that give rise strictly to bone, cartilage, and marrow stroma in mice and humans32,33. SSCs are distinct from mesenchymal stromal cells which represent highly heterogenous mixtures of cell types57. We’ve since have leveraged our knowledge of SSCs to understand degenerative skeletal conditions including osteoarthritis, non-unions and age-related osteoporosis46,58,59. More recently, we used our isolation methods to discover tissue resident SSCs within mouse and human IVDs. In this proposal, the overarching objective is to identify the intrinsic cues that dictate SSC fate into IVD tissue as well as examine the extrinsic cues that may guide IVD regeneration using a novel microfracture surgical model in the mouse caudal IVD. Our preliminary data suggests that IVD SSCs are distinctly more chondrogenic than femur SSCs in their differentiation capacity both in vitro and in vivo. Additionally, we found that mouse IVD SSCs exclusively express HOXA4 and their skeletal fate decisions can be dictated by the addition of morphogens FGF2, SHH and WNT3A in vitro. We also found that acute microfracture injury of caudal IVDs in the mouse tail does not amplify resident IVD SSCs and transplantation of IVD SSCs into microfractured IVD fail to generate fibrocartilage, thus suggesting they may require the guidance of additional factors for cartilage differentiation in vivo. In this proposal, our overall hypothesis is that HOXA4 is an intrinsic regulator, while FGF2 is an extrinsic regulator of IVD tissue fate, and that modulating these pathways in IVD SSCs can be used as a potential stem cell-targeting therapy for combating IVD degeneration. In Aim 1, we will address this hypothesis by modulating HOXA4 expression in IVD and femur (control) SSCs via lentiviral transduction and subsequently perform in vitro and in vivo differentiation assays to assess their cartilagenic IVD output. In Aim 2, we will test if the injection of morphogens FGF2, SHH and WNT3A can change the fate decision of microfractured resident SSCs to regenerate damaged IVD tissue in vivo. Ultimately, this set of basic and pre-clinical proposed experiments will further define the concept the SSC diversity and set the foundation for the clinical translation of stem cell-based therapies for preventing and reversing IVD-related musculoskeletal diseases.